This program is engaged in defining cellular and molecular mechanisms of transplant tolerance. It is based on the widely accepted premise that some cellular populations are toleragenic or can be made so via expression of key immunomodulatory molecules or when given with adjunctive treatments to modulate the immune response. We have sought improvements of methods used to generate transduced lymphocyte or embryonic stem cell populations (manuscript submitted)and methods to enhance expression of retroviral or lentiviral transgenes in lymphocyte populations (manuscript in press). Our projects are now in the phase of testing these methods and transduced cell populations in vivo for their ability to induce immune tolerance, or restore cell populations from embryonic progenitors. Our program is also examining the fate of the memory T cell response following exposure to sub-lethal irradiation. This knowledge should contribute to our ability to respond to radiologic terrorism, as well as potentially define a mechanism by which memory responses to autoantigen can be terminated. Induction of Tolerance by the Restricted Expression of Alloantigen. This project was designed to generate "veto" cells which eliminate cytolytic effector cells specific for MHC alloantigen. MHC class I Dd was fused to a potent signaling molecule, the zeta (z) chain of the TCR and expressed on lymphocytes in transgenic mice. Transgenic mice expressing high copy numbers of the Dd-zeta fusion molecule have markedly diminished numbers of CD4+ and CD8+ T cells and a reciprocally high percentage of CD3-CD5+ cells which are TCR-CD4-CD8- cells but which are of T cell lineage as they express Thy 1.1,CD44, and Ly6C. Interestingly they express supraphysiologic level of Dd. These cells appear similar to memory CD8+ CTL, not only by cell surface appearance, but also functionally, as they have abundant cytolytic activity which can be activated by 1) TCR hybridomas recognizing cognate peptide in the context of Dd 2) mAb to Dd, and 3)cellular recognition of Dd by spleen cells from FVB mice primed to Dd. These cells potently kill cells that bind to Dd such as EL-4 cells which bind Dd through cell surface Ly49A. The mechanism by which CTL activity is mediated is being investigated, but does not appear to depend on Fas ligand expression or TRAIL expression. Furthermore, these memory CTL like cells also potently block the development of an anti-Dd CTL response by both naive and primed FVB lymphocytes. Thus, these CD3-CD5+ T lineage cells represent an ideal type of a "veto" cell in being able to diminish even potent memory Dd specific CTL responses, yet because they lack TCR, they also lack the ability to induce GVHD. This work has recently been published in the journal "Blood". We are currently studying whether IL-15 can skew development of thymocytes into the veto cell lineage by mating the Dd-zeta mice to IL-15 transgenics. Additional studies are underway in which primary CD8+ T cells and/or NK cell populations will be transfected with Dd-z using a lentiviral vector. The veto activity of such cells will then be tested in an in vivo skin allograft rejection model. Alteration of retroviral vectors for targeted expression in vivo. Retroviral vectors have been used in clinical gene therapy trials to effect permanent cure of inborn errors of metabolism, such as ADA-SCID, because these vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells. Current retroviral vectors used in clinical trials lack a selectable marker (neo) gene, to avoid immune responses to transfected cells. As a result, selection of transfected packaging cell lines has become an extremely labor intensive process involving the screening of thousands of clones by RNA analyses. We first addressed the task of facilitating selection of transfected packaging cell lines by adopting the strategy of using a selectable marker, but placing it outside of the inter-LTR regions of the retroviral construct. Thus, the selection marker is active in the packaging cells, but is not incorporated into the retroviral transcript produced by the packaging cell lines. We created a novel viral construct (MPSV-GFP)in which a neo gene selection marker was directly cloned into the plasmid backbone of MPSV-GFP, yielding (MPSV-GFP)-neo. We have demonstrated that this is an effective strategy in that within three weeks of transfection, 45% of packaging cells transfected with (MPSV-GFP)-neo and raised on a high concentration of G418, expressed the construct, compared to only 8% of packaging cells that were co-transfected with separate MPSV-GFP and neo constructs and produced a higher (2-3 fold) viral titre. We have further demonstrated that the selection marker is not present in target cells transfected with retrovirus produced by the(MPSV-GFP)-neo packaging cell lines, indicating that tandem repeats were not incorporated in the retroviral transcript. An invention report is being pursued by patent attorneys and a manuscript submitted for publication. The second project, in which we attempted to boost expression of a retroviral transgene in lymphocytes has revealed that the murine 3' light chain enhancer (mE3')has locus control region (LCR) activity in both reducing variability of expression and enhancing expression in T cell lines, primary T cells, and B cell lines. A manuscript covering this work has been accepted in Human Gene Therapy. In vivo expression patterns mediated by the mE3' are being pursued by transducing cells with the current retroviral vector, but also testing a novel lentiviral vector into which the mE3' has been cloned. Immunologic responses to murine embryonic stem cells and committed progenitors. Embryonic stem (ES) cells have the potential to differentiate into progenitor cells that are committed to a given lineage. There has been an explosion of interest in using ES cells, or their committed progenitor offspring, to repair or replace damaged tissues, yet the immune response to such cells across histocompatibility barriers has been poorly defined. This project examined the immmune response to ES cells and their committed progenitor offspring. The goal is to genetically engineer ES cells to inhibit graft rejection while providing tissue for repair. ES cells, embryoid body cells (EB) and neural progenitor cells (NP) fail to express MHC molecules and so are killed by NK cells in both syngeneic and allogeneic mice with lysis highly dependent on LFA-1/ICAM interactions. However, the response in syngeneic mice is weak and injection of ES cells into syngeneic mice yields tumor cells which progress and kill the mice. In contrast, embryonal carcinoma cells (EC), which arise in syngeneic animals injected with ES cell populations are not killed by NK or LAK cells. The failure to kill EC cells appears to relate in some EC cells to lack of expression of ICAM and in others, an increased expression of both classical and non-classical class I MHC molecules. The mechanism by which EC cells develop from ES cells is also being explored. Because of the tumorigenic potential of ES cells, prevention of rejection cannot be aimed at ES cells, but will instead be aimed at non-tumorigenic committed progentior or mature differentiated cells. To protect such committed or differentiated cells from rejection, our strategy is to clone protective molecules, such as Fas-ligand, IL-10 or TGF-beta into ES cells and place them under control of a lineage specific promoter, so that they will not be expressed until the cell assumes a differentiated (non-malignant) phenotype. Over the past year, we have been exploring whether lentiviral vectors, equipped with neural specific promoters, can mediate high levels of expression in NP cells. Using such vectors, we will explore whether healing of CNS lesions can be facilitated by such cells in a murine model of multiple sclerosis (EAE). Additionally, we may clone into an ES cell line a suicide gene expressing an enzyme under control of an ES cell specific promoter. Effects of Sub-Lethal Irradiation on the Memory Immune Response to Biological Agents. This project explores whether the memory T cell response to vaccine antigens is affected by sublethal irradiation, such as might be experienced by a proportion of persons in detonation of a nuclear device or "dirty bomb". Mice will be immunized with listeria monocytogenes to generate a memory immune response that has been shown to be protective against a challenge. Following establishment of immunity, the mice will be exposed to varying doses of sub-lethal irradiation (200-750R), and then challenged a short time later with live listeria. Control immunized non-irradiated mice should survive such challenge, while this dose should be lethal to non-immunized mice. Studies are now underway. This project incorporates FY2002 projects 1Z01BN002027-02, 1Z01BN002028-02, and 1Z01BN002038-04.